Antimicrobial compounds

ABSTRACT

The present invention relates to a compound of formula (I) AA-AA-AA-R 1 -R 2  (I) wherein, in any order, 2 of said AA (amino acid) moieties are cationic amino acids and 1 of said AA is an amino acid with a lipophilic R group, the R group having 14-27 non-hydrogen atoms; R 1  is a N atom, which may be substituted by a branched or unbranched C 1 -C 10  alkyl or aryl group, which group may incorporate up to 2 heteroatoms selected from N, O and S; and R 2  is an aliphatic moiety having 2-20 non-hydrogen atoms, said moiety being linear, branched or cyclic. The invention further relates to formulations containing these compounds, solid supports having these compounds attached thereto, the use of these compounds in therapy, particularly as antimicrobial, anti-tumour or anti-biofilm agents and non-therapeutic uses of these compounds, particularly their use in inhibiting biofilm formation or removing a biofilm.

The present invention relates to peptides and similar molecules whichexhibit antimicrobial activity, in particular which exhibit thatactivity against microorganisms existing as a biofilm.

Peptides and their derivatives have long been recognised astherapeutically interesting molecules. A wide variety of organisms usepeptides as part of their host defence mechanism. Antimicrobial peptideshave been isolated from species as diverse as bacteria and mammals.Generally, these peptides have a net positive charge and a propensity toform amphiphilic α-helix or β-sheet structures upon interaction with theouter phospholipid bilayer in bacterial cell membranes. In most casesthe detailed molecular mechanisms of the antibiotic action are unknown,although some peptides categorised as class L (lytic) peptides arebelieved to interact with bacterial cell membranes, probably formingion-channels or pores.

The majority of known antibacterial peptides comprise 10 or more,typically 20 or more amino acids, this number of amino acid beingrequired in order to provide sufficient length for the peptide,generally in α-helical form, to span the bacterial cell membrane andform a pore. Such a mechanism is the generally accepted way in which themajority of such peptides exert their cytotoxic activity.

Synthesis of the antibacterial peptides of the prior art can bedifficult, and typically requires the peptides to be synthesised bybacteria or other organisms which can be cultured and harvested to yieldthe peptide of interest, additional processing steps after isolation ofthe direct product of translation are generally required. If activepeptides could be identified which were shorter, this would enableeconomic manufacture by synthesis from the amino acid building blocks oravailable di- or tri-peptides. In addition, short peptides would offeradvantages for biodelivery. There is a growing demand for antibioticswhich can be administered without the need for an injection, such as byinhalation and absorption across the blood capillaries of the nasalpassages.

The search for novel antibiotics has taken on particular urgency becauseof the increasing number of bacterial strains which are exhibitingresistance to known and extensively used drugs. Those operating in thefields of medicine as well as agriculture, environmental protection andfood safety are constantly requiring new antibacterial agents and mayhave to treat a given population or site with several differentantibacterial agents in order to effectively combat the undesirablebacteria.

A biofilm is a collection, or community, of microorganisms surrounded bya matrix of extracellular polymers (also known in the art as aglycocalyx). These extracellular polymers are typically polysaccharides,notably polysaccharides produced by the organisms themselves, but theycan contain other biopolymers as well. A biofilm will typically beattached to a surface, which may be inert or living, but it has alsobeen observed that biofilms may form from microorganisms attached toeach other or at any interface. Such a mode of growth is protective tothe microorganisms, and renders them difficult to remove or eradicate.Biofilms cause significant commercial, industrial and medical problems,in terms of infections, contamination, fouling and spoilage etc.

Formation of a biofilm typically begins with the attachment offree-floating microorganisms to a surface. These first colonists mayadhere to the surface initially through weak, reversible van der Waalsforces. If the colonists are not immediately separated from the surface,they can anchor themselves more permanently using cell adhesionstructures such as pili.

The first colonists typically facilitate the arrival of other cells byproviding more diverse adhesion sites and beginning to build the matrixthat holds the biofilm together. Some species are not able to attach toa surface on their own but are often able to anchor themselves to thematrix or directly to earlier colonists. During this colonization thecells are able to communicate via quorum sensing. Once colonization hasbegun, the biofilm may grow through a combination of cell division andrecruitment. The final stage of biofilm formation is known asdevelopment, and is the stage in which the biofilm is established andmay only change in shape and size. This development of biofilm allowsfor the cells to become more antibiotic resistant. A biofilm in the“development” stage may be referred to as a “mature” biofilm.

The microorganisms in a biofilm community display properties at thecellular level (phenotype) that are not shared by their planktonic(free-floating) equivalents. It is believed that such sessilemicroorganisms are profoundly different from planktonic free-floatingcells. Further differences can be also be observed at the communitylevel and are attributed to the effects of the extracellular matrix.Perhaps most notable is the commonly observed phenomenon thatmicroorganisms in a biofilm environment do not display the samesusceptibilities to anti-microbial agents, e.g. antibiotics, antifungalsand microbicides, and host immune defences or clearance mechanisms. Itis thought that this resistance is due to the barrier effect of theextracellular matrix and/or a phenotypic change in the microbesthemselves. It is also believed that microorganisms in biofilms may growmore slowly, and as a result take up anti-microbial agents more slowly.

There exists a need therefore for new antimicrobial agents which areeffective antibiofilm agents, particularly in clinical contexts.

The present inventors have now identified a small group of modifiedpeptides which exhibit an impressive set of characteristics, includinggood antimicrobial activity and low toxicity and specifically a goodactivity against microorganisms which are present in biofilms.

Thus in one aspect is provided a compound, preferably a peptide, offormula (I)

AA-AA-AA-R₁-R₂  (I)

wherein, in any order, 2 of said AA (amino acid) moieties are cationicamino acids, preferably lysine or arginine but may be histidine or anynon-genetically coded or modified amino acid carrying a positive chargeat pH 7.0, and 1 of said AA is an amino acid with a large lipophilic Rgroup, the R group having 14-27 non-hydrogen atoms and preferablycontaining 2 or more, e.g. 2 or 3, cyclic groups which may be fused orconnected, these cyclic groups will typically comprise 5 or 6non-hydrogen atoms, preferably 6 non-hydrogen atoms (in the case offused rings of course the non-hydrogen atoms may be shared);

R₁ is a N atom, which may be but preferably is not substituted by abranched or unbranched C₁-C₁₀ alkyl or aryl group, e.g. methyl, ethyl orphenyl, and this group may incorporate up to 2 heteroatoms selected fromN, O and S;

R₂ is an aliphatic moiety having 2-20 non-hydrogen atoms, preferablythese are carbon atoms but oxygen, nitrogen or sulphur atoms may beincorporated, preferably R₂ comprises 3-10, most preferably 3-6non-hydrogen atoms and the moiety may be linear, branched or cyclic. Ifthe R₂ group comprises a cyclic group this is preferably attacheddirectly to the nitrogen atom of R₁.

Preferred compounds of the invention incorporate an R₂ group which islinear or branched, in particular a linear or branched alkyl groupincluding ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl and isomers thereof, hexyl and isomers thereof etc.;propyl, isopropyl, butyl and isobutyl are especially preferred.

Of the R₂ groups which comprise a cyclic group, preferred are moleculesin which R₂ is cyclohexyl or cyclopentyl.

Suitable non-genetically coded amino acids and modified amino acidswhich can provide a cationic amino acid include analogues of lysine,arginine and histidine such as homolysine, ornithine, diaminobutyricacid, diaminopimelic acid, diaminopropionic acid and homoarginine aswell as trimethylysine and trimethylornithine,4-aminopiperidine-4-carboxylic acid,4-amino-1-carbamimidoylpiperidine-4-carboxylic acid and4-guanidinophenylalanine.

The large lipophilic R group may contain hetero atoms such as O, N or Sbut typically there is no more than one heteroatom, preferably it isnitrogen. This R group will preferably have no more than 2 polar groups,more preferably none or one, most preferably none.

In a preferred embodiment the compounds, preferably peptides, of theinvention are of formula (II)

AA₁-AA₂-AA₁-R₁-R₂  (II)

wherein:

AA₁ is a cationic amino acid, preferably lysine or arginine but may behistidine or any non-genetically coded or modified amino acid carrying apositive charge at pH 7.0;

AA₂ is an amino acid with a large lipophilic R group, the R group having14-27 non-hydrogen atoms and preferably containing 2 or more, e.g. 2 or3, cyclic groups which may be fused or connected, these cyclic groupswill typically comprise 5 or 6 non-hydrogen atoms, preferably 6non-hydrogen atoms; and

R₁ and R₂ are as defined above.

Further compounds of the invention include compounds of formulae (III)and (IV):

AA₂-AA₁-AA₁-R₁-R₂  (III)

AA₁-AA₂-AA₂-R₁-R₂  (IV)

wherein AA₁, AA₂, R₁ and R₂ are as defined above. However molecules offormula (II) are preferred.

As discussed further below, compounds of the invention may be peptidemimetics, in particular with linkages other than amide bonds betweensaid AA moieties, nevertheless, amide linkages are preferred.

From amongst the above compounds certain are preferred. In particular,compounds wherein the amino acid with a large lipophilic R group,conveniently referred to herein as AA₂, is tributyl tryptophan (Tbt) ora biphenylalanine derivative such as Phe (4-(2′-naphthyl)), Phe(4-(1′-naphthyl)), Phe(4-4′-n-butylphenyl), Phe (4-4′-biphenyl) orPhe(4-4′-t-butylphenyl); Phe (4-(2′-naphthyl)) and Tbt being mostpreferred.

A further preferred group of compounds are those in which —R₁-R₂together is selected from the group consisting of —NHCH(CH₃)₂,—NH(CH₂)₅CH₃, —NH(CH₂)₃CH₃, —NH(CH₂)₂CH₃, —NHCH₂CH(CH₃)₂, —NHcyclohexyland —NHcyclopentyl, most preferred are compounds in which —R₁-R₂ is thegroup —NHCH(CH₃), or —NH(CH₂)₅CH₃.

The compounds include all enantiomeric forms, both D and L amino acidsand enantiomers resulting from chiral centers within the amino acid Rgroups and R₂.

From hereon when referring to amino acids, standard single-letter aminoacid abbreviations or standard three-letter amino acid codes may beused.

Preferred compounds are those of table 1 herein and their equivalents inwhich Arg residues have been replaced with alternative cationicresidues, e.g., Lys.

Most preferred compounds are Compounds 1 and 2 in the Examples hereinand their equivalents incorporating other cationic residues in place ofArg.

In a further aspect is provided compounds of formulae (I), (II), (III)or (IV) for use in therapy, particularly for use as an antimicrobial(e.g. antibacterial) agent, but also as an anti-tumour agent. Thecompounds of the invention are also for use in treatingbiofilm-associated infections.

Such antimicrobial molecules also have non-therapeutic uses, for examplein agriculture or in domestic or industrial situations as sterilisingagents for materials susceptible to microbial contamination. Thus, in afurther aspect, the present invention provides the use of the compoundsof the invention as antimicrobial, particularly as antibacterial agents.Biofilms are known to cause problems in environmental settings and thepresent invention further provides a method of inhibiting biofilmformation or removing a biofilm which comprises contacting said biofilmwith a compound of the invention.

A ‘biofilm-associated infection’ is a microbial infection of a subjectwhere it is known or suspected that the microbes are present as abiofilm. Typically it will be an infection where the existence of abiofilm is relevant to the clinical condition, e.g. to the diagnosis orprognosis, to the treatment regimen, to the severity of the infection,to the duration of the infection up to the point of treatment oranticipated in the future. ‘Treatment’ includes prophylactic treatmentand encompasses a reduction in size of the biofilm, a reduction innumber of living microorganisms within the biofilm and prevention orreduction in the tendency of microorganisms within the biofilm to breakfree and form new biofilm colonies. Treatment includes an improvement,observed by clinician or patient, in one or more of the symptomsassociated with the infection.

The size, structure, integrity, and number of microbes in a biofilm canbe analysed by any convenient method. For instance, scanning andtransmission electronic microscopy is often used to asses the size,integrity and structure of a biofilm. Analysis of biofilm is describedin the Examples hereto.

The biofilms that may be treated in accordance with the invention arenot limited in terms of the microorganisms they contain, the lyticmolecules described herein target the cell membranes and therefore havea fairly non-specific activity. Accordingly, the biofilm may compriseany class, genus or species of microorganism, namely any microorganismthat may form a biofilm. Such microorganisms typically include bacteria,including any genus or species of bacteria. Thus, the bacteria may begram positive or gram negative, or gram test non-responsive. They may beaerobic or anaerobic. The bacteria may be pathogenic or non-pathogenic.

It is particularly surprising that the molecules defined herein are ableto kill bacteria in mature biofilms and the treatment of such biofilmsis particularly preferred.

The biofilm may comprise Gram positive bacteria, Pseudomonas aeruginosaand/or fungi. Biofilms comprising or consisting of Gram positivebacteria are preferred targets.

Biofilms comprising Staphylococcus are preferred targets, with biofilmscomprising S. haemolyticus being especially preferred.

Biofilms may also contain fungi, algae and other organisms such asparasitic protozoa. Mixed colony biofilms are known and treatableaccording to the methods described herein.

Chronic wounds are discussed above and are a preferred therapeutictarget, these include diabetic foot ulcers, venous leg ulcers andpressure ulcers as well as surgical wounds (postoperative woundinfections) which have become chronic.

Medical devices are a particular class of surface on which a biofilm mayform and represent a further preferred therapeutic target according tothe present, invention.

This may include any kind of line, including catheters (e.g. centralvenous and urinary catheters), prosthetic devices e.g., heart valves,artificial joints, false teeth, dental crowns, dental caps and softtissue implants). Any kind of implantable (or “in-dwelling”) medicaldevice is included (e.g. stents, intrauterine devices, pacemakers,intubation tubes, prostheses or prosthetic devices, lines or catheters).An “in-dwelling” medical device may include a device in which any partof it is contained within the body, i.e. the device may be wholly orpartly in-dwelling.

In specific embodiments of the invention the peptides andpeptidomimetics may be used in the treatment of native valveendocarditis, acute otitis media, chronic bacterial prostatitis,pneumonia, dental plaque, periodontitis, biofilm infections inrespiratory diseases, which may include cystic fibrosis, and devicerelated infection associated with implantable or prosthetic medicaldevices e.g. prosthetic valve endocarditis or infection of lines orcatheters or artificial joints or tissue replacements.

The wounds may be acute or chronic. Acute wounds are wounds that proceedorderly through the three recognised stages of the healing process (i.e.the inflammatory stage, the proliferative stage and the remodellingphase) without a protracted time course. Chronic wounds, however, arethose wounds that do not complete the ordered sequence of biochemicalevents because the wound has stalled in one of the healing stages.Viewed alternatively a chronic wound is a wound that has not healedwithin at least 40 days, preferably at least 50 days, more preferably atleast 60 days, most preferably at least 70 days.

The wound to be treated may be a breach in, or denudement of, the tissuefor instance caused by surgical incision or trauma, e.g., mechanical,thermal, electrical, chemical or radiation trauma; a spontaneouslyforming lesion such as a skin ulcer (e.g. a venous, diabetic or pressureulcer); a blister (e.g. a friction or thermal blister, or a blistercaused by pathogen infection such as chicken pox); an anal fissure or amouth ulcer.

The treatment of chronic wounds represents a particularly preferredaspect of the present invention.

Although biofilms are now more widely recognised as contributing tomedical conditions, they are also implicated in non-medical problemscaused by microbial colonisation of surfaces. This may be, for example,in domestic, industrial, research or hospital settings where surfacesneed to be kept free of bacterial contamination.

As noted above the biofilm may be present on a surface. The surface isnot limited and includes any surface on which a microorganism may occur,particularly, as noted above, a surface exposed to water or moisture.The surface may be biotic or abiotic, and inanimate (or abiotic)surfaces include any such surface which may be exposed to microbialcontact or contamination. Thus particularly included are surfaces onmachinery, notably industrial machinery, or any surface exposed to anaquatic environment (e.g. marine equipment, or ships or boats or theirparts or components), or any surface exposed to any part of theenvironment e.g. pipes or on buildings. Such inanimate surfaces exposedto microbial contact or contamination include in particular any part of:food or drink processing, preparation, storage or dispensing machineryor equipment, air conditioning apparatus, industrial machinery e.g. inchemical or biotechnological processing plants, storage tanks andmedical or surgical equipment. Any apparatus or equipment for carryingor transporting or delivering materials, which may be exposed to wateror moisture is susceptible to biofilm formation. Such surfaces willinclude particularly pipes (which term is used broadly herein to includeany conduit or line). Representative inanimate or abiotic surfacesinclude, but are not limited to food processing, storage, dispensing orpreparation equipment or surfaces, tanks, conveyors, floors, drains,coolers, freezers, equipment surfaces, walls, valves, belts, pipes, airconditioning conduits, cooling apparatus, food or drink dispensinglines, heat exchangers, boat hulls or any part of a boat's structurethat is exposed to water, dental waterlines, oil drilling conduits,contact lenses and storage cases.

Thus in a further aspect the present invention provides a method ofinhibiting biofilm formation or removing a biofilm which comprisescontacting said biofilm with a peptide or peptidomimetic as definedherein. Said biofilm may be on any of the surfaces described above.

The term “contacting” encompasses any means of delivering the peptide orpeptidomimetic to the biofilm, whether directly or indirectly, and thusany means of applying the peptide or mimetic to the biofilm or exposingthe biofilm to the peptide or mimetic e.g. applying the peptide ormimetic directly to the biofilm.

Optionally, the compounds of the present invention may be attached to asolid support, for example in order to prevent the colonization ofbacteria thereon. Thus, in addition to their use in the sterilisation ofcontaminated surfaces, the present compounds can be attached to surfacesin order to prevent their contamination. In particular, the presentcompounds can be attached to solid supports in order to inhibit theformation of a biofilm thereon. In the context of the present invention,the terms “solid support” and “surface” are interchangeable.

Thus, in a further aspect is provided a solid support is provided havingattached thereto a compound of the present invention. Such solidsupports include but are not limited to the surfaces described above.Surfaces on which biofilms can form include medical devices, containers,carriers or ducts carrying water or other fluids etc. Medical devicesare a particular class of surface on which a biofilm may form andrepresent a preferred surface onto which the compounds of the presentinvention can be attached.

The term “medical devices” includes any kind of line, includingcatheters (e.g. central venous and urinary catheters), prostheticdevices e.g., heart valves, artificial joints, false teeth, dentalcrowns, dental caps and soft tissue implants). Any kind of implantable(or “in-dwelling”) medical device is included (e.g. stents, intrauterinedevices, pacemakers, intubation tubes, prostheses or prosthetic devices,lines or catheters). An “in-dwelling” medical device may include adevice in which any part of it is contained within the body, i.e. thedevice may be wholly or partly in-dwelling.

The compounds of the present invention can be attached to solid supportsby any means known in the art. The compounds may be directly orindirectly attached to the solid support, i.e. they may be attached by alinking group. Preferably the molecules are directly attached to thesolid support although, as discussed below, modification of thecompounds of the invention may be required to permit attachment.Typically, the compounds are covalently attached to a desired solidsupport. Therefore, optionally the compounds of the present inventioncomprise a chemical group which permits covalent attachment to saidsolid support. Alternatively, the compounds are modified to permitcovalent attachment to said solid support.

The term “modified” or “modification” includes the replacement of achemical group of a compound of the invention with a chemical groupwhich permits covalent attachment to said support. The term alsoincludes the further substitution of an existing chemical group with achemical group which permits covalent attachment to said support. Theterm also includes the scenario where, rather than replacing or furthersubstituting a chemical group of a pre-existing compound of the presentinvention, a compound of the invention is designed to comprise achemical group which permits covalent attachment to said solid support,and is prepared in this form. Preferably said modification results inthe introduction of no more than 5, preferably no more than 3, morepreferably no more than 2 non-hydrogen atoms.

The exact nature of the chemical group will depend on the chemicalnature of the desired surface to which the compound is to be attached.Likewise, the surface of the solid support may be modified to enableattachment. A variety of suitable chemical groups are well-known in theart and the appropriate groups for attachment would be readilydeterminable by the skilled man. By way of example only, chemical groupswhich permit covalent attachment to surfaces may beheteroatom-containing groups, including oxygen-containing groups such ascarboxyl groups, nitrogen-containing groups such as amide groups andsulphur-containing groups such as thiol groups. Covalent bonds which canexist between the compounds of the invention and the desired supportsinclude but are not limited to ether, ester, amide, amine, sulfide,thioether and thioester bonds. Thus, for example, an ester link may havebeen formed from an alcohol moiety on the support and a carboxylic acidmoiety within the molecule of the invention or vice versa.Alternatively, covalent attachment of the molecules of the invention tosurfaces may be achieved via connections which do not involveheteroatoms, for instance via alkene-vinyl or vinyl-vinyl groupconnections, wherein either the alkene or vinyl group is within themolecule of the invention and the other necessary group is on a desiredsurface. Cycloaddition reactions may also be used to covalently attachthe molecules of the invention to a desired surface.

Preferably, said covalent attachment is between the R₂ group of thecompound and the support. Thus, preferably —R₂ comprises or is modifiedto comprise, a chemical group which permits covalent attachment to thedesired support.

When the compounds of the present invention are attached to a desiredsolid support via a covalent bond between —R₂ and the support, —R₂preferably comprises a carboxyl group, said carboxyl group permittingcovalent attachment to the support. More preferably, in the form as itis attached to the solid support, —R₁-R₂ is selected from the groupconsisting of —NHCH(CH₃)CO—, —NH(CH₂)₅CO—, —NH(CH2)₃CO—, —NH(CH₂)₂CO—and —NHCH₂CH(CH₃)CO—, and most preferably R₁R₂ is —NHCH(CH₃)CO— or—NH(CH₂)₅CO—

The molecules exhibit antimicrobial activity, in particular they exert acytotoxic effect through a direct membrane-affecting mechanism and canbe termed membrane acting antimicrobial agents. These molecules arelytic, destabilising or even perforating the cell membrane. This offersa distinct therapeutic advantage over agents which act on or interactwith proteinaceous components of the target cells, e.g. cell surfacereceptors. While mutations may result in new forms of the targetproteins leading to antibiotic resistance, it is much less likely thatradical changes to the lipid membranes could occur to prevent thecytotoxic effect. The lytic effect causes very rapid cell death and thushas the advantage of killing bacteria before they have a chance tomultiply. In addition, the molecules may have other useful propertieswhich kill or harm the target microbes e.g. an ability to inhibitprotein synthesis, thus they may have multi-target activity.

Thus in a further aspect is provided the molecules of the invention foruse in destabilising and/or permeabilising microbial cell membranes. By‘destabilising’ is meant a perturbation of the normal three dimensionallipid bi-layer configuration including but not limited to membranethinning, increased membrane permeability (typically not involvingchannels) to water, ions or metabolites etc. which also impairs therespiratory systems of the bacteria.

When the molecules are used against a biofilm they are preferably watersoluble and R₂ has only 2-6, preferably 2-4 non-hydrogen atoms.Antifungal applications favour molecules in which R₂ has 6-20, e.g. 6-15non-hydrogen atoms, preferably 7-20, e.g. 7-15 non-hydrogen atoms.

β and γ amino acids as well as a amino acids are included within theterm ‘amino acids’, as are N-substituted glycines which may all beconsidered AA units. The molecules of the invention include betapeptides and depsipeptides.

The compounds of formulae (I) to (IV) may be peptidomimetics andpeptidomimetics of the peptides described and defined herein are afurther aspect of the present invention. A peptidomimetic is typicallycharacterised by retaining the polarity, three dimensional size andfunctionality (bioactivity) of its peptide equivalent but wherein thepeptide bonds have been replaced, often by more stable linkages. By‘stable’ is meant more resistant to enzymatic degradation by hydrolyticenzymes. Generally, the bond which replaces the amide bond (amide bondsurrogate) conserves many of the properties of the amide bond, e.g.conformation, steric bulk, electrostatic character, possibility forhydrogen bonding etc. Chapter 14 of “Drug Design and Development”,Krogsgaard, Larsen, Liljefors and Madsen (Eds) 1996, Horwood Acad. Pubprovides a general discussion of techniques for the design and synthesisof peptidomimetics. In the present case, where the molecule is reactingwith a membrane rather than the specific active site of an enzyme, someof the problems described of exactly mimicking affinity and efficacy orsubstrate function are not relevant and a peptidomimetic can be readilyprepared based on a given peptide structure or a motif of requiredfunctional groups. Suitable amide bond surrogates include the followinggroups: N-alkylation (Schmidt, R. et al., Int. J. Peptide Protein Res.,1995, 46, 47), retro-inverse amide (Chorev, M and Goodman, M., Acc.Chem. Res, 1993, 26, 266), thioamide (Sherman D. B. and Spatola, A. F.J. Am. Chem. Soc., 1990, 112, 433), thioester, phosphonate,ketomethylene (Hoffman, R. V. and Kim, H. O. J. Org. Chem., 1995, 60,5107), hydroxymethylene, fluorovinyl (Allmendinger, T. et al.,Tetrahydron Lett., 1990, 31, 7297), vinyl, methyleneamino (Sasaki, Y andAbe, J. Chem. Pharm. Bull. 1997 45, 13), methylenethio (Spatola, A. F.,Methods Neurosci, 1993, 13, 19), alkane (Lavielle, S. et. al., Int. J.Peptide Protein Res., 1993, 42, 270) and sulfonamido (Luisi, G. et al.Tetrahedron Lett. 1993, 34, 2391).

The peptidomimetic compounds of the present invention will typicallyhave 3 identifiable sub-units which are approximately equivalent in sizeand function to amino acids (AA units). The term ‘amino acid’ may thusconveniently be used herein to refer to the equivalent sub-unit of apeptidomimetic compound. Moreover, peptidomimetics may have groupsequivalent to the R groups of amino acids and discussion herein ofsuitable R groups and of N and C terminal modifying groups applies,mutatis mutandis, to peptidomimetic compounds.

As is discussed in the text book referenced above, as well asreplacement of amide bonds, peptidomimetics may involve the replacementof larger structural moieties with di- or tripeptidomimetic structuresand in this case, mimetic moieties involving the peptide bond, such asazole-derived mimetics may be used as dipeptide replacements.Peptidomimetics and thus peptidomimetic backbones wherein the amidebonds have been replaced as discussed above are, however, preferred.

Suitable peptidomimetics include reduced peptides where the amide bondhas been reduced to a methylene amine by treatment with a reducing agente.g. borane or a hydride reagent such as lithium aluminium-hydride. Sucha reduction has the added advantage of increasing the overallcationicity of the molecule.

Other peptidomimetics include peptoids formed, for example, by thestepwise synthesis of amide-functionalised polyglycines. Somepeptidomimetic backbones will be readily available from their peptideprecursors, such as peptides which have been permethylated, suitablemethods are described by Ostresh, J. M. et al. in Proc. Natl. Acad. Sci.USA (1994) 91, 11138-11142. Strongly basic conditions will favourN-methylation over O-methylation and result in methylation of some orall of the nitrogen atoms in the peptide bonds and the N-terminalnitrogen.

Preferred peptidomimetic backbones include polyesters, polyamines andderivatives thereof as well as substituted alkanes and alkenes. Thepeptidomimetics will preferably have N and C termini which may bemodified as discussed herein.

The invention provides methods of treating microbial infections byadministering the various molecules described herein. Likewise methodsof destabilising microbial cell membranes are provided. The amountadministered should be effective to kill all or a proportion of thetarget microbes or to prevent or reduce their rate of reproduction orotherwise to lessen their harmful effect on the body. The clinician orpatient should observe improvement in one or more of the parameters orsymptoms associated with the infection. Administration may also beprophylactic.

In a further aspect is provided a method of producing a compound of theinvention.

The peptides of the invention may be synthesised in any convenient way.Generally the reactive groups present (for example amino, thiol and/orcarboxyl) will be protected during overall synthesis. The final step inthe synthesis will thus be the deprotection of a protected derivative ofthe invention.

In building up the peptide, one can in principle start either at theC-terminal or the N-terminal although the C-terminal starting procedureis preferred.

Methods of peptide synthesis are well known in the art but for thepresent invention it may be particularly convenient to carry out thesynthesis on a solid phase support, such supports being well known inthe art.

A wide choice of protecting groups for amino acids are known andsuitable amine protecting groups may include carbobenzoxy (alsodesignated Z) t-butoxycarbonyl (also designated Boc),4-methoxy-2,3,6-trimethylbenzene sulphonyl (Mtr) and9-fluorenylmethoxy-carbonyl (also designated Fmoc). It will beappreciated that when the peptide is built up from the C-terminal end,an amine-protecting group will be present on the α-amino group of eachnew residue added and will need to be removed selectively prior to thenext coupling step.

Carboxyl protecting groups which may, for example be employed includereadily cleaved ester groups such as benzyl (Bzl), p-nitrobenzyl (ONb),pentachlorophenyl (OPClP), pentafluorophenyl (OPfp) or t-butyl (OtBu)groups as well as the coupling groups on solid supports, for examplemethyl groups linked to polystyrene.

Thiol protecting groups include p-methoxybenzyl (Mob), trityl (Trt) andacetamidomethyl (Acm).

A wide range of procedures exists for removing amine- andcarboxyl-protecting groups. These must, however, be consistent with thesynthetic strategy employed. The side chain protecting groups must bestable to the conditions used to remove the temporary α-amino protectinggroup prior to the next coupling step.

Amine protecting groups such as Boc and carboxyl protecting groups suchas tBu may be removed simultaneously by acid treatment, for example withtrifluoroacetic acid. Thiol protecting groups such as Trt may be removedselectively using an oxidation agent such as iodine.

References and techniques for synthesising peptidomimetic compounds andthe other bioactive molecules of the invention are described herein andthus are well known in the art.

Formulations comprising one or more compounds of the invention inadmixture with a suitable diluent, carrier or excipient constitute afurther aspect of the present invention. Such formulations may be for,inter alia, pharmaceutical (including veterinary) or agriculturalpurposes or for use as sterilising agents for materials susceptible tomicrobial contamination, e.g. in the food industry. Suitable diluents,excipients and carriers are known to the skilled man.

The peptides defined herein exhibit broad antimicrobial activity andthus are also suitable as antiviral and antifungal agents, which willhave pharmaceutical and agricultural applications, and as promoters ofwound healing or spermicides. All of these uses constitute furtheraspects of the invention. As discussed above, use an antibiofilm agentsis a preferred use of the compounds of the invention.

Methods of treating or preventing bacterial, viral or fungal infectionsor of treating tumours which comprises administration to a human oranimal patient one or more of the peptides or peptidomimetics as definedherein constitute further aspects of the present invention.

The compositions according to the invention may be presented, forexample, in a form suitable for oral, nasal, parenteral, intravenal,intratumoral or rectal administration.

As used herein, the term “pharmaceutical” includes veterinaryapplications of the invention:

The active compounds defined herein may be presented in the conventionalpharmacological forms of administration, such as tablets, coatedtablets, nasal sprays, solutions, emulsions, liposomes, powders,capsules or sustained release forms. The peptides are particularlysuitable for topical administration, e.g. in the treatment of diabeticulcers. Conventional pharmaceutical excipients as well as the usualmethods of production may be employed for the preparation of theseforms. Tablets may be produced, for example, by mixing the activeingredient or ingredients with known excipients, such as for examplewith diluents, such as calcium carbonate, calcium phosphate or lactose,disintegrants such as corn starch or alginic acid, binders such asstarch or gelatin, lubricants such as magnesium stearate or talcum,and/or agents for obtaining sustained release, such ascarboxypolymethylene, carboxymethyl cellulose, cellulose acetatephthalate, or polyvinylacetate.

The tablets may if desired consist of several layers. Coated tablets maybe produced by coating cores, obtained in a similar manner to thetablets, with agents commonly used for tablet coatings, for example,polyvinyl pyrrolidone or shellac, gum arabic, talcum, titanium dioxideor sugar. In order to obtain sustained release or to avoidincompatibilities, the core may consist of several layers too. Thetablet-coat may also consist of several layers in order to obtainsustained release, in which case the excipients mentioned above fortablets may be used.

Organ specific carrier systems may also be used.

Injection solutions may, for example, be produced in the conventionalmanner, such as by the addition of preservation agents, such asp-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are thenfilled into injection vials or ampoules.

Nasal sprays which are a preferred method of administration may beformulated similarly in aqueous solution and packed into spraycontainers either with an aerosol propellant or provided with means formanual compression. Capsules containing one or several activeingredients may be produced, for example, by mixing the activeingredients with inert carriers, such as lactose or sorbitol, andfilling the mixture into gelatin capsules.

Suitable suppositories may, for example, be produced by mixing theactive ingredient or active ingredient combinations with theconventional carriers envisaged for this purpose, such as natural fatsor polyethyleneglycol or derivatives thereof.

Lotions, creams, solutions, gels and ointments etc. are preferred dosageforms. When R, is a linear aliphatic group it is preferably formulatedas an ointment or other fatty formulation. When R, is a branchedaliphatic moiety it is preferably formulated in an aqueous solution, gelor cream.

Dosage units containing the active molecules preferably contain 0.1-10mg, for example 1-5 mg of the antimicrobial agent. The pharmaceuticalcompositions may additionally comprise further active ingredients,including other cytotoxic agents such as other antimicrobial peptides.Other active ingredients may include different types of antibiotics,cytokines e.g. IFN-γ, TNF, CSF and growth factors, immunomodulators,chemotherapeutics e.g. cisplatin or antibodies.

The bioactive molecules, when used in topical compositions, aregenerally present in an amount of at least 0.1%, by weight. In mostcases, it is not necessary to employ the peptide in an amount greaterthan 1.0%, by weight.

In employing such compositions systemically (intra-muscular,intravenous, intraperitoneal), the active molecule is present in anamount to achieve a serum level of the bioactive molecule of at leastabout 5 μg/ml. In general, the serum level need not exceed 500 μg/ml. Apreferred serum level is about 100 μg/ml. Such serum levels may beachieved by incorporating the bioactive molecule in a composition to beadministered systemically at a dose of from 1 to about 10 mg/kg. Ingeneral, the molecule(s) need not be administered at a dose exceeding100 mg/kg.

Methods of treating environmental or agricultural sites or products, aswell as foodstuffs and sites of food production, or surfaces or toolse.g. in a hospital environment with one or more of the molecules of theinvention to reduce the numbers of viable bacteria present or limitbacterial growth or reproduction or to reduce, disrupt, inhibit orotherwise weaken a biofilm constitute further aspects of the presentinvention.

The invention will now be further described with reference to thefollowing non-limiting Examples and Figures, in which

FIG. 1 are graphs showing the effect of 24 h treatment with rifampicin,linezolid, tetracycline and vancomycin on 24 h old biofilm of 6different staphylococcal strains. For each strain, bars represent fromleft to right: negative control, positive control, treatment withantibiotic (vancomycin, linezold, tetracycline) concentration 5 mg/L, 50mg/L, and 500 mg/L. For rifampicin, the concentrations were 0.01 mg/L,0.1 mg/L and 1 mg/L. Values are means of three experiments±SD. * meansstrong suppression of metabolic activity. ** means complete suppressionof metabolic activity.

FIG. 2 are graphs showing the effect of 24 h treatment with 2 differentSAMPs on 24 h old biofilm of 6 different staphylococcal strains. Foreach strain, bars represent from left to right negative control,positive control, treatment with SAMPs in concentration 5 mg/L, 50 mg/L,and 500 mg/L. Values are means of three experiments±SD. * means strongsuppression of metabolic activity. ** means complete suppression ofmetabolic activity.

FIG. 3 are photos showing 48 h old S. haemolyticus 51-07 biofilm grownon cover slide discs. The biofilms were stained with LIVE/DEAD stainingand investigated with confocal laser scanning microscopy. Untreatedbiofilm (A); biofilm treated for 24 h with vancomycin 50 mg/L (B);vancomycin 500 mg/L (C); tetracycline 50 mg/L (D); tetracycline 500 mg/L(E); Compound 1 50 mg/L (F) and Compound 1 500 mg/L (G).

FIG. 4 is a graph showing the effect of one day topical treatmentagainst S. aureus FDA486 in a murine skin infection model. Each mousewas treated at 9 am, 12 noon and 3 pm. The skin biopsy was collected at6 pm. The median value is shown.

FIG. 5 is a graph showing the effect of one day topical treatmentagainst Streptococcus pyogenes CS301 in a murine skin infection model.Each mouse was treated at 7 am, 10 am and 1 pm. The skin biopsy wascollected at 4 pm. The median value is shown.

FIG. 6 is a graph showing the effect of one day topical treatmentagainst S. aureus FDA486 in a murine skin infection model. Each mousewas treated at 9 am, 12 noon and 3 pm. The skin biopsy was collected at6 pm. The median value is shown.

FIG. 7 is a photomicrograph of A) Uncoated aminomethylated polystyreneHL particles B) Uncoated aminomethylated polystyrene HL particles after24 h incubation with Staphylococcus epidermidis C)Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl-polystyreneparticles after 24 h incubation with Staphylococcus epidermidis D)Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl-aminohexanoyl-polystyreneparticles after 24 h incubation with Staphylococcus epidermidis.

EXAMPLES Example 1 Preparation and Physical, Antimicrobial andHaemolytic Properties of Molecules of the Invention Peptide SynthesisChemicals

Protected amino acids Boc-Arg-OH, and Boc-4-phenyl-Phe were purchasedfrom Bachem AG while Boc-4-iodophenylalanine was purchased from Aldrich.isopropylamine, propylamine, hexylamine, butylamine, hexadecylamine,isobutylamine, cyclohexylamine and cyclopentylamine making up theC-terminal of the peptide were purchased from Fluka.Diisopropylethylamine (DIPEA), 1-hydroxybenzotriazole (1-HOBt),chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP) andO-(benzotriazol-1-yl)-N,N,N′,N′ tetramethyluronium hexafluorophosphate(HBTU) were purchased from Fluka. 4-n-Butylphenylboronic acid,4-t-butylphenylboronic acid, 4-biphenylboronic acid, 2-napthylboronicacid, tri ortho-tolylphosphine, benzylbromide and palladium acetate werepurchased from Aldrich. Solvents were purchased from Merck, Riedel-deHaën or Aldrich.

Preparation of Amino Acids

Preparation of Boc-2,5,7-tri-tert-butyl tryptophan-OH:

A mixture of H2N-Trp-OH (1.8 g, 8.8 mmol), t-BuOH (4.7 g, 63.4 mmol) intrifluoroacetic acid (19 mL) is stirred at 70° C. for 3 hours. Thevolume of the resulting mid-brown translucent solution is reduced on arotary evaporator at room temperature for 30 min and then triturated bymeans of adding 60 mL of 7% (by weight) NaHCO₃ drop-wise. The gray/whitegranular solid obtained is then recovered by vacuum filtration and driedin vacuo at room temperature for 24 hours. The product is isolated bycrystallization from a near boiling mixture of 40% ethanol in water.Volumes typically are approximately 20 mL per gram of crude product.

A first crystallization from crude produces isolated product of 80-83%purity (HPLC) with respect to all other substances in the sample andapproximately 94-95% purity with respect to the known TBT analogues.Yields at this stage are in the range 60-0.65%.

Benzylation of Boc-4-iodophenylalanine.

Boc-4-iodophenylalanine (1 equivalent) was dissolved in 90% methanol inwater and neutralized by addition of caesium carbonate until a weakalkaline pH (determined by litmus paper). The solvent was removed byrotary evaporation, and remaining water in the caesium salt ofBoc-4-iodophenylalanine was further reduced by repeated azeotropicdistillation with toluene. The resulting dry salt was dissolved indimethylformamide (DMF), benzylbromide (1.2 equivalents) was added andthe resulting mixture was stirred for 6-8 h. At the end of the reactionDMF was removed under reduced pressure and an oil containing the titlecompound is formed. This oil was dissolved in ethyl acetate and theresulting solution was washed with equal volumes of citric acid solution(three times), sodium bicarbonate solution and brine. The title compoundwas isolated as a pale yellow oil in 85% yield by flash chromatographyusing dichloromethane:ethyl acetate (95:5) as eluent. Crystalline benzylBoc-4-iodophenylalanine could be obtained by recrystallisation fromn-heptane.

General Procedure for Suzuki Couplings:

Benzyl Boc-4-iodophenylalanine (1 equivalent), arylboronic acid (1.5equivalents), sodium carbonate (2 equivalents), palladium acetate (0.05equivalent) and tri ortho-tolylphosphine (0.1 equivalent) was added to adegassed mixture of dimethoxyethane (6 ml/mmol benzylBoc-4-iodophenylalanine) and water (1 ml/mmol benzylBoc-4-iodophenylalanine). The reaction mixture was kept under argon andheated to 80° C. for 4-6 h. After cooling to room temperature themixture is filtered through a short pad of silica gel and sodiumcarbonate. The filter cake was further washed with ethyl acetate. Thefiltrates were combined and the solvents were removed under reducedpressure. The products were isolated by flash chromatography usingmixtures of ethyl acetate and n-hexane as eluent.

Preparation of Boc-Phe(4-4′-biphenyl)-OBn:

The title compound was prepared in 61% yield from 4-biphenylboronic acidusing the general procedure for Suzuki couplings.Boc-Phe(4-4′-biphenyl)-OBn was isolated by recrystallisation of thecrude product from n-heptane.

Preparation of Boc-Phe(4-(2′-Naphtyl))-OBn:

The title compound was prepared in 68% yield from 2-naphtylboronic acidusing the general procedure for Suzuki couplings.Boc-Phe(4-(2′-Naphtyl))-OBn was isolated by recrystallisation of thecrude product from n-heptane.

General Procedure for Deesterification of Benzyl Esters:

The Benzyl ester is dissolved in DMF and hydrogenated for 2 days atambient pressure using 10% Pd on carbon as catalyst. At the end of thereaction the catalyst is removed by filtration and the solvent isremoved under reduced pressure. The free acids are isolated byrecrystallisation from diethyl ether.

Preparation of Boc-Phe(4-4′-biphenyl)-OH:

The title compound was prepared in 61% yield fromBoc-Phe(4-4′-biphenyl)-OBn using the general procedure fordeesterification.

Preparation of Boc-Phe(4-(2′-Naphtyl))-OH:

The title compound was prepared in 68% yield fromBoc-Phe(4-(2-Naphtyl))-OBn using the general procedure fordeesterification.

General Procedure for Solution Phase Peptide Synthesis Using HBTU.

The peptides were prepared in solution by stepwise amino acid couplingusing Boc protecting strategy according to the following generalprocedure. The C-terminal peptide part with a free amino group (1 eq)and the Boc protected amino acid (1.05 eq) and 1-hydroxybenzotriazole(1-HOBt) (1.8 eq) were dissolved in DMF (2-4 ml/mmol amino component)before addition of diisopropylethylamine (DIPEA) (4.8 eq). The mixturewas cooled on ice and O-(benzotriazol-1-yl)-N,N,N′,N′ tetramethyluroniumhexafluorophosphate (HBTU) (1.2 eq) was added. The reaction mixture wasshaken at ambient temperature for 1-2 h. The reaction mixture wasdiluted by ethyl acetate and washed with citric acid, sodium bicarbonateand brine. The solvent was removed under vacuum and the Boc protectinggroup of the resulting peptide was deprotected in the dark using 95% TFAor acetylchloride in anhydrous methanol.

Solution phase amide formation using PyCloP. Synthesis ofBoc-Arg-N(CH₂Ph)₂. A solution of Boc-Arg-OH (1 eq), NH(CH₂Ph)₂ (1.1 eq)and PyCloP (1 eq) in dry DCM (filtered through alumina) (2 ml) and DMF(1 ml). The solution was cooled on ice and DIPEA (2 eq) was added understirring. The solution was stirred for 1 h at room temperature. Thereaction mixture was evaporated, and redissolved in ethyl acetate andwashed with citric acid, sodium bicarbonate and brine. The solvent wasremoved under vacuum and the Boc protecting group of the resultingpeptide was deprotected in the dark using 95% TFA.

Peptide Purification and Analysis.

The peptides were purified using reversed phase HPLC on a Delta-Pak(Waters) C₁₈ column (100 Å, 15 μm, 25×100 mm) with a mixture of waterand acetonitrile (both containing 0.1% TFA) as eluent. The peptides wereanalyzed by RP-HPLC using an analytical Delta-Pak (Waters) C₁₈ column(100 Å, 5 μm, 3.9×150 mm) and positive ion electrospray massspectrometry on a VG Quattro quadrupole mass spectrometer (VGInstruments Inc., Altringham, UK).

TABLE 1 Compounds of the invention General compound formula:Arg-AA₂-Arg-R₁-R₂ Purity Compound AA₂ R₁R₂ (HPLC) 1 2,5,7-tri-tert-NHCH(CH₃)₂ butyltryptophan 2 2,5,7-tri-tert- NH(CH₂)₅CH₃ butyltryptophan3 2,5,7-tri-tert- NH(CH₂)₃CH₃ 87 butyltryptophan 4 2,5,7-tri-tert-NH(CH₂)₂CH₃ 99 butyltryptophan 5 2,5,7-tri-tert- NH(CH₂)₁₅CH₃ 80butyltryptophan 6 2,5,7-tri-tert- NHCH₂CH(CH₃)₂ 97 butyltryptophan 72,5,7-tri-tert- NHcyclohexyl 95 butyltryptophan 8 2,5,7-tri-tert-NHcyclopentyl 91 butyltryptophan 9 Phe(4-4′- NHCH(CH₃)₂ biphenyl) 10Phe(4-4′- NH(CH₂)₅CH₃ biphenyl) 11 Phe(4-(2′- NHCH(CH₃)₂ Naphtyl)) 12Phe(4-(2′- NH(CH₂)₅CH₃ Naphtyl))

Antimicrobial Assay

MIC determinations on Staphylococcus aureus, strain ATCC 25923,Methicillin resistant Staphylococcus aureus (MRSA) strain ATCC 33591 andMethicillin resistant Staphylococcus epidermidis (MRSE) strain ATCC27626 were performed by Toslab AS using standard methods. Amsterdam, D.(1996) Susceptibility testing of antimicrobials in liquid media, inAntibiotics in Laboratory Medicine. 4th ed (Lorian, V., Ed.) pp 75-78,Williams and Wilkins Co, Baltimore.

TABLE 2 Antimicrobial and toxic properties of compounds of the inventionC. albicans S. aureus MRSA MRSE S. pyogenes E. coli P. aeruginosaCompound (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) EC50 1 25 <2<2 <2 <2 7 7 720 2 5 2 2 <1 2 5 5 32 3 10 2 3 <2 2 350 4 10 2 3 <2 2 6205 >100 5 4 4 6 >100 >100 38 6 10 <2 3 2 2 300 7 10 <2 2 2 <2 55 8 10<2 >15 <2 2 340

Example 2 In Vitro Broad Panel Screening of Selected Molecules of theInvention 2.0 Materials and Methods 2.1 Antimicroblals

Vials of pre-weighed Compound 1 and Compound 2 were supplied by LytixBiopharma AS.

General compound formula: AA₁-AA₂-AA₁-R₁R₂ AA₁ AA₂ R₁R₂ Compound 1 Arg2,5,7-tri-tert- NHCH(CH₃)₂ butyltryptophan Compound 2 Arg2,5,7-tri-tert- NH(CH₂)₅CH₃ butyltryptophan

2.2 Bacterial Isolates

Bacterial isolates used in this study were from various sourcesworldwide stored at GR Micro Ltd. and maintained, with minimalsub-culture, deep frozen at −70° C. as a dense suspension in a highprotein matrix of undiluted horse serum. The species used and theircharacteristics are listed in Table 2. These included 54 Gram-positivebacteria, 33 Gram-negative bacteria and 10 fungi.

2.3 Determination of Minimum Inhibitoy Concentration (KIC)

MICs were determined using the following microbroth dilution methods forantimicrobial susceptibility testing published by the Clinical andLaboratory Standards Institute (CLSI, formerly NCCLS):

M7-A6 Vol. 23 No. 2. January 2003 Methods for Dilution AntimicrobialSusceptibility Tests for Bacteria that Grow Aerobically; ApprovedStandard-Sixth Edition.

M100-S15 Vol. 25 No 1. January 2005 Performance Standards forAntimicrobial Susceptibility Testing; Fifteenth InformationalSupplement.

M11-A6 Vol. 24 No. 2. Methods for Antimicrobial Susceptibility Testingof Anaerobic Bacteria; Approved Standard-Sixth Edition.

M27-A2 Vol. 22 No. 15. Reference Method for Broth Dilution AntifungalSusceptibility Testing of Yeasts; Approved Standard-Second Edition.

M38-A Vol. 22 No. 16. Reference Method for Broth Dilution AntifungalSusceptibility Testing of Filamentous Fungi; Approved Standard.

MIC estimations were performed using wet plates, containing theantibacterials or antifungals, prepared at GR Micro Ltd.

Cation-adjusted Mueller-Hinton broth (Oxoid Ltd., Basingstoke, UK andTrek Diagnostic Systems Ltd., East Grinstead, UK) (supplemented with 5%laked horse blood for Streptococcus spp., Corynebacterium jeikeium andListeria monocytogenes) was used for aerobic bacteria, with an initialinoculum of approximately 10⁵ colony-forming units (CFU)/mL.

Haemophilus test medium (Mueller-Hinton broth containing 0.5% yeastextract and Haemophilus test medium supplement which contains 15 mg/L ofeach of haematin and NAD, all obtained from Oxoid Ltd., Basingstoke, UK)was used for the Haemophilus influenzae and inoculated withapproximately 10⁵ CFU/mL.

Supplemented Brucella broth (SBB) was used for the anaerobic strainswith an inoculum of approximately 10⁵ CFU/mL. SBB is a broth consistingof 1% peptone, 0.5% ‘Lab-lemco’, 1% glucose and 0.5% sodium chloridesupplemented with 5 μg/L haemin and 1 μg/L vitamin K (both obtained fromSigma Aldrich Ltd.)

Yeast and filamentous fungal MIC were performed in MOPS buffered RPMI1640 medium (MOPS buffer obtained from Sigma Aldrich Ltd., RPMI 1640obtained from Invitrogen Ltd, Paisley, Scotland). The yeast inocula werein the range 7.5×10²-4×10³ CFU/mL and the filamentous fungiapproximately 8×10³-1×10⁵ CFU/mL

Following normal practice all the plates containing Mueller-Hinton brothwere prepared in advance, frozen at −70° C. on the day of preparationand defrosted on the day of use. Fungal, Haemophilus and anaerobic MICdeterminations were all performed in plates prepared on the same day.

3.0 Results

The results are shown in Table 3 as a single line listing.

The MIC data obtained is very encouraging and indicates that thepeptides have quite a broad spectrum of activity.

TABLE 3 Single line list of the in vitro activity of two novelantimicrobial peptides against a panel of Gram-positive bacteria,Gram-negative bacteria and fungi. Compound Compound Species andproperties 1 (mg/L) 2 (mg/L) Candida albicans ATCC90028 - referencestrain 32 4 Candida albicans ATCC24433 - reference strain 64 8 Candidatropicalis ATCC750 - reference strain 4 4 Candida parapsilosisATCC90018 - reference strain 64 8 Candida (Issatchenkia) kruseiATCC6258 - reference strain 8 32 Aspergillus niger - G. R. Microcollection 32 4 Trichophyton mentagrophytes - G. R. Micro collection 8 4Trichophyton interdigitale - G. R. Micro collection 16 4 Microsporumcanis - G. R. Micro collection 16 4 Cryptococcus neoformans - G. R.Micro collection 8 2 Escherichia coli ATCC25922 - antibiotic-susceptibletype strain 32 4 Escherichia coli ATCC32518 - β-lactamase positive typestrain 32 8 Escherichia coli - multi-drug resistant clinical isolate 328 Klebsiella aerogenes NCTC11228 - antibiotic-susceptible type strain 648 Klebsiella aerogenes - multi-drug resistant clinical isolate 32 8Enterobacter sp - antibiotic-susceptible clinical isolate 64 8Enterobacter sp - multi-drug resistant clinical isolate ≥128 8Pseudomonas aeruginosa ATCC27853 - antibiotic-susceptible type strain 328 Pseudomonas aeruginosa - multi-drug resistant clinical isolate 8 4Stenotrophomonas maltophilia - antibiotic-susceptible clinical isolate32 4 Salmonella sp - antibiotic-susceptible clinical isolate 16 8Salmonella sp - multi-drug resistant clinical isolate 16 8 Shigella sp -antibiotic-susceptible clinical isolate 32 4 Morganella morganii -multi-drug resistant clinical isolate 32 8 Haemophilus influenzae -β-lactamase negative clinical isolate 32 16 Haemophilus influenzae -β-lactamase positive clinical isolate 16 4 Haemophilus influenzaeβ-lactamase negative ampicillin-resistant clinical isolate 16 8Moraxella catarrhalis - β-lactamase positive clinical isolate 4 16Moraxella catarrhalis - reduced fluoroquinolone susceptibility clinicalisolate 8 16 Acinetobacter baumanii - antibiotic-susceptible clinicalisolate 64 16 Staphylococcus aureus ATCC 29213 - antibiotic-susceptiblecontrol strain 8 4 Staphylococcus aureus ATCC 25923 -antibiotic-susceptible control strain 8 4 Staphylococcus aureus ATCC43300 - methicillin-resistant control strain 8 4 Staphylococcus aureus -methicillin-resistant clinical isolate 8 4 Staphylococcus aureus -multi-drug-resistant clinical isolate 16 4 Staphylococcus aureus -teicoplanin-intermediate clinical isolate 16 4 Staphylococcusepidermidis antibiotic susceptible clinical isolate 4 8 Staphylococcusepidermidis methicillin-resistant clinical isolate 4 2 Staphylococcushaemolyticus - antibiotic susceptible clinical isolate 4 4Staphylococcus saprophyticus - antibiotic susceptible clinical isolate 20.5 Enterococcus faecalis - ATCC 29212 antibiotic-susceptible controlstrain 16 4 Enterococcus faecalis vancomycin-susceptible clinicalisolate 32 4 Enterococcus faecalis vancomycin-resistant (VanA) clinicalisolate 32 4 Enterococcus faecalis vancomycin-resistant (VanB) clinicalisolate ≥128 8 Enterococcus faecalis high-level gentamicin-resistantclinical isolate 64 8 Enterococcus faecium vancomycin-susceptibleclinical isolate 16 4 Enterococcus faecium vancomycin-resistant (VanA)clinical isolate 32 4 Enterococcus faecium vancomycin-resistant (VanB)clinical isolate 16 4 Enterococcus gallinarum vancomycin-resistant(VanC) clinical isolate 8 4 Streptococcus pneumoniae - ATCC 49619antibiotic-susceptible control strain 32 16 Streptococcus pneumoniae -penicillin-susceptible clinical isolate 32 8 Streptococcus pneumoniae -penicillin-intermediate clinical isolate 32 16 Streptococcuspneumoniae - penicillin-resistant clinical isolate 32 16 Streptococcuspneumoniae - multi-drug resistant clinical isolate 32 16 Streptococcuspyogenes - Macrolide (MLS) resistant clinical isolate 16 8 Streptococcuspyogenes - Macrolide (M-type) resistance clinical isolate 16 8Corynebacterium jeikeium - antibiotic-susceptible clinical isolate 8 4Corynebacterium jeikeium - multi-drug resistant clinical isolate 8 2Listeria monocytogenes - antibiotic-susceptible clinical isolate 16 8MU50 Staphylococcus aureus (MRSA) - VISA type strain 16 4 EMRSA3Staphylococcus aureus (MRSA) - SSCmec type 1 8 4 EMRSA16 Staphylococcusaureus (MRSA) - SSCmec type 2 16 4 EMRSA1 Staphylococcus aureus (MRSA) -SSCmec type 3 16 4 EMRSA15 Staphylococcus aureus (MRSA) - SSCmec type 48 4 HT2001254 Staphylococcus aureus (MRSA) - PVL positive 8 4Streptococcus agalactiae - antibiotic-susceptible clinical isolate 8 8Streptococcus agalactiae - macrolide-resistant clinical isolate 16 8Group C Streptococcus - antibiotic-susceptible clinical isolate 16 8Group C Streptococcus - macrolide-resistant clinical isolate 32 16 GroupG Streptococcus - antibiotic-susceptible clinical isolate 16 8 Group GStreptococcus - macrolide-resistant clinical isolate 16 8 Streptococcusmitis - antibiotic-susceptible clinical isolate 32 16 Streptococcusmitts - macrolide-resistant clinical isolate 64 16 Streptococcusconstellatus - antibiotic-susceptible clinical isolate 64 16Streptococcus constellatus - macrolide-resistant clinical isolate 32 16Streptococcus oralis - antibiotic-susceptible clinical isolate 64 16Streptococcus oralis - macrolide-resistant clinical isolate 64 16Streptococcus bovis - antibiotic-susceptible clinical isolate 32 8Streptococcus bovis - macrolide-resistant clinical isolate 8 2Streptococcus sanguis - antibiotic-susceptible clinical isolate 32 16Streptococcus sanguis - macrolide-resistant clinical isolate 32 16Clostridium perfringens - antibiotic-susceptible clinical isolate ≥12832 Clostridium difficile - antibiotic-susceptible clinical isolate 64 32Propionibacterium acnes - antibiotic-susceptible clinical isolate 4Propionibacterium acnes - antibiotic-resistant clinical isolate 2

Example 3 In Vitro Efficacy Against Biofilm Material and MethodsBacterial Strains and Growth Conditions

The clinical strains used in this study are listed in Table 4.

TABLE 4 Bacterial strains used in this study; susceptibility toantibiotics and SAMPs, and biofilm profile. Biofilm MIC antibiotics(mg/L) MIC SAMP molecules optical Strain Source RIF VAN TET LZD GEN OXACompound 1 Compound 2 Ica^(d) density SH^(a) TUH Blood <0.016 4 1 0.564 >256 8 4 + 0.37 51-03 culture SH TUH Blood 0.016 2 0.5 0.5 64 >256 84 + 0.77 51-07 culture SE^(b) TUH Blood 0.016 2 2 2 256 16 4 2 + 0.6308-16 culture SE RP62A Blood <0.016 4 0.5 1 8 8 8 4 + 1.33 ATCC 35984culture SA^(c) PIA 9 Joint <0.016 2 0.5 2 1 1 8 4 + 3.20 fluid SA PIA90Joint 0.016 2 0.5 1 0.5 1 8 2 + 0.40 fluid ^(a)SH; Staphylococcushaemolyticus ^(b)SE; Staphylococcus epidermidis ^(c)SA; Staphylococcusaureus ^(d)ica; PCR detection of icaD as a marker of the operon

Six staphylococcal strains (2 S. epidermidis, 2 S. haemolyticus and 2 S.aureus) were selected based on their previously known biofilm formingcapacity. Bacteria were grown overnight at 37° C. in cation adjustedMueller-Hinton II Broth (MHIIB).

Antibiotics, SAMPs and Susceptibility Testing Under Planktonic GrowthCondition

We determined the MICs of oxacillin, gentamicin, tetracycline,vancomycin and linezolid using E-test (AB Biodisk, Solna, Sweden) andMICs of rifampicin using broth microdilution assay. Breakpoints wereinterpreted according to EUCAST criteria. The MIC values for Compound 1and 2 determined with broth microdilution assay.

Biofilm Formation and Quantification of Activity Against Biofilms

Biofilm formation was induced in 96-well flat bottom microtitre plates(Nunclon Surface, NUNC). First, overnight cultures were diluted 1:100 inMHIIB (S. epidermidis and S. haemolyticus) or tryptic soy broth (TSB)with 5% glucose and 5% NaCl (S. aureus). 200 μl of this bacterialsuspension (10⁷ cfu/ml) was added to each well and incubated for 24 h at37° C. After 24 h the wells were carefully washed twice withphosphate-buffered saline (PBS) to remove planktonic bacteria. Thewashing procedure was carefully evaluated by measuring metabolicactivity of the PBS with the Alamar blue method, described in detailbelow.

The washed biofilms were subjected to treatment with antibiotics orSAMPs at different concentrations. Stock solutions of the tetracycline(Sigma Aldrich), vancomycin (Alpharma) and linezolid (Pfizer) werediluted in MHIIB to 5 mg/L, 50 mg/L and 500 mg/L, and rifampicin (SigmaAldrich) was diluted in MHIIB to 0.01 mg/L, 0.1 mg/L and 1 mg/L.Trifluoroacetate salts of the SAMPs were dissolved in sterile water anddiluted to 5 mg/L, 50 mg/L and 500 mg/L in MHIIB. 200 μl of antibioticsor Compound 1 or 2, in different concentrations, were added to each welland incubated for 24 h at 37° C. Positive controls were untreatedbiofilms only added 200 μl MHIIB. Negative controls were only 200 μlMHIIB, with no bacteria added.

The metabolic activity of the biofilm was quantified with a slightlymodified method previously described by Pettit et al. Antimicrob. AgentsChemother. 2005; 49: 2612-7. Briefly, after the 24 h incubation withantimicrobial agents the wells were again washed twice with PBS and thenadded 250 μl MHIIB with 5% Alamar blue (AB; Biosource, Camarillo,Calif., USA). AB is a redox indicator which both fluoresces and changescolour in response to chemical reduction. The extent of reduction is areflection of bacterial cell viability. After 1 h incubation at 37° C.,absorbance was recorded at 570 and 600 nm using Versamax tuneablemicroplate reader (Molecular Devices, Sunnyvale, Calif., USA). Allassays were performed 3 times with 8 parallels. The highest and lowestvalue of each run was excluded from the analyses, and the remaining 18values were averaged.

The biofilm method quantifying metabolic activity was compared to astandard semiquantitative biomass-quantification method in 96-wellmicrotitre plates. For these experiments we grew 24 h biofilms of all 6staphylococcal strains and analyzed metabolic activity with AB, asdescribed above. Biomass quantification on the 24 h biofilms wasperformed by staining the biofilm with crystal violet (CV). Afterstaining, ethanol:acetone (70:30) was added to each well to dissolveremaining crystal violet along the walls of the wells. The opticaldensity (OD) was then recorded at 570 nm using a spectrophotometer.

Biofilm Imaging

One ml aliquots of MHIIB-diluted overnight culture was used to grow S.haemolyticus TUH 51-07 biofilm on plastic coverslides (Thermanox,cellculture treated on one side, NUNC) in 24-well dishes (Falcon 3047,Becton Dickinson, N.J., USA) for 24 h. The coverslides were then washedcarefully with PBS, moved to a new plate and treated for 24 h withtetracycline 50 mg/L and 500 mg/L, vancomycin 50 mg/L and 500 mg/L, orCompound 1 50 mg/L and 500 mg/L. The coverslides were washed again with9% NaCl and stained with a LIVE/DEAD kit (Invitrogen Molecular Probes,Eugene, Oreg., USA) following the manufacturer's instructions. Thisstain contains SYTO 9 (green fluorescent) and propidium iodide (PI; redfluorescent), both binding to DNA. When used alone, the SYTO 9 generallystains all bacteria in a population; both those with intact and thosewith damaged membranes. In contrast, PI penetrates only bacteria withdamaged membranes, causing a reduction in the SYTO 9 stain greenfluorescence when both dyes are present. We examined treated anduntreated biofilms with a Leica TCS SP5 (Leica Microsystems CMS Gmbh,Mannheim, Germany) confocal laser scanning microscope (CLSM). Imageswere obtained using a 63×1.2 NA HCX PL APO water immersion lens. Fordetection of SYTO9 (green channel), we used the 488 nm line of the argonlaser and a detection bandwidth of 495-515 nm. For PI detection (redchannel), we used the 561 nm line and a detection bandwidth of 615-660run. The two fluorescent signals were collected sequentially at 400 Hz.Image analyses and export was performed in Leica LAS AF version 1.8.2.

Statistical Analysis and Evaluations

The percent reduction of AB was calculated according to themanufacturer's formula (Trek Diagnostic System). We calculated mean andstandard deviations (SD) of all repeated measurements. Pearson'stwo-tailed correlation between the AB method and the CV method wascalculated on averaged data from all 6 staphylococcal strains.Statistical analysis was performed with SPSS for Windows softwareversion 14.0.

In FIGS. 1 and 2 we present the crude percentage values of AB reduction,including positive and negative control. We defined two levels ofantimicrobial suppression of metabolic activity. A strong suppressionwas obtained if an agent, after adjusting for the negative control, at acertain concentration caused ≥75% reduction of AB compared to positivecontrol. A complete suppression was obtained if an agent at a certainconcentration caused a reduction of AB≤negative control value+2SD.

As expected, the untreated biofilm showed green cells with intact cellmembrane FIG. 3a . In the biofilm subjected to treatment with Compound 1in a concentration of 50 mg/L and especially 500 mg/L almost all cellsare stained red, indicating dead bacteria (FIGS. 3 f and g). In biofilmsubjected to treatment with 500 mg/L tetracycline a significant part ofthe cells are still green indicating live bacteria with intact cellmembrane (FIG. 3e ). Treatment of the biofilm with vancomycin (FIG. 3d )at a concentration close to the peak values obtained in clinicalpractice (50 mg/L) showed predominantly green cells (live organisms).

Example 4 In Vivo Activity of Compound 1 and 2

The skin of mice was infected with Staphylococcus aureus orStreptococcus pyogenes and subsequently given a total of threetreatments at three hourly intervals. Three hours after the lasttreatment, skin biopsies were collected and the number of colony formingunits (CFUs) present in the skin sample was determined.

Results are shown in FIGS. 4, 5 and 6 expressed as the number of colonyforming units per mouse.

In experiment 1 (FIG. 4), compound 1 was applied to the murine skin aspart of either a cream or a gel containing 2% (w/w) of compound 1. Thesame cream or gel without compound 1 was used as a negative control(placebo). Bactroban 2% cream was used as a positive control. It canclearly be seen that the number of CFUs was reduced when a cream or gelcontaining compound 1 was applied to the murine skin, compared to thenegative control, indicating that compound 1 exerted an antimicrobialeffect against Staphylococcus aureus. The efficacy of standard clinicaltreatment, Bactroban 2% cream, had no significant effect under thetreatment regime. The nature of the carrier, cream or gel, had nosignificant effect.

In experiment 2 (FIG. 5), compound 1 was applied in two differentconcentrations, as either a 1% or a 2% gel. A placebo gel and a knownantibacterial “bactroban (mupericin)” were used as controls. It can beseen that gels containing compound 1 were more effective at reducing thenumber of CFUs from a Streptococcus pyogenes CS 301 infection than theplacebo gel or the bactroban. The gel containing 2% of compound 1 wasmore effective than the gel containing only 1% of compound 1.

In experiment 3 (FIG. 6), compound 2 was applied in a 2% creamformulation on a Staphylococcus aureus FDA 486 infection in the murineskin infection model. A placebo cream and two known antibacterials,“Fucidin (fucidic acid) ointment 2%” and “Bactroban (mupericin) cream2%” were used as controls. It can be seen that a cream containingcompound 2 was more effective at reducing the number of CFUs than theplacebo and fucidin or bactroban.

Example 5 Peptidic Surface Modification of Polystyrene ParticlesPreparation of Coated Particles

5.1

To a 20 mL peptide reactor was added 560 mg (0.5 mmole) ofaminomethylated polystyrene HL particles (100-200 mesh, 0.90 mmole/gsubstitution) which were then washed 2×10 min with 8 mL DCM. A further 8mL of DCM was then added and the particles permitted to swell for 1 hourbefore the reactor was drained prior to the first coupling.

5.2

To 8 mL of DMF was added 3 equivalents of Boc amino-acid and 683 mg (3.6equivalents) of O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium(HBTU) coupling reagent. Immediately prior to transfer of the mixture tothe reactor 0.855 mL (10 equivalents) of N-Ethyldiisopropylamine (DIPEA)was added and the mixture transferred to the reaction in one portion.The reactor was then agitated moderately whilst the reaction waspermitted to run overnight at room temperature.

3.

The particles were then washed 3×15 min with 8 mL DMF and 2×10 min with8 mL DCM.

4.

At this point a small sample was removed from the reactor and subjectedto the Kaiser test in order to determine whether there was any remaininguncoupled amine.

5.

If the Kaiser test gives a positive result the procedure was repeatedfrom and including point 2 with the same amino acid. In the event thatthe test was negative (no uncoupled amine remaining) 8 mL of TFA/DCM(1:1) was added to the reactor to remove the Boc group from the newlycoupled amino acid and the reactor agitated moderately for 1 hour.

6.

The particles were then washed 3×15 min with 8 mL DCM and 2×10 min with8 mL DMF.

7.

The procedure was now repeated from and including point 2 to andincluding point 6 with the next amino acid to be coupled.

8.

When the final amino acid unit has completed stage 5 in the procedureoutlined above the particles were washed 4×15 min with 8 mL DCM anddried in the reactor under nitrogen flow for 30 min before being driedunder vacuum at room temperature for 24 hours. The particles were thenstored in sealed vials at 4° C.

In the manner described above, the following peptide coated particleswere prepared in close to quantitative yields using the appropriateamino acids:

Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl-polystyrene(control peptide)

Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl-aminohexanoyl-polystyrene(peptide according to the invention)

Reduced Bacterial Colonization of Surface Coated Particles

The particles prepared above were incubated with Staphylococcusepidermidis for 24 h. The amount of bacterial colonization on thebacterial surface was determined by fluorescence microscopy (excitationfrequency of 485 nm, emission frequency 498 nm) after staining of thebiofilm forming bacteria by Syto9 according to standard procedures.

The effect on colonization was determined by visual inspection ofphotomicrographs of the polystyrene particles.

FIG. 7 below shows the photomicrograph of:

A) Uncoated aminomethylated polystyrene HL particles

B) Uncoated aminomethylated polystyrene HL particles after 24 hincubation with Staphylococcus epidermidis

C) Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl-polystyreneparticles after 24 h incubation with Staphylococcus epidermidis

D)Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl-aminohexanoyl-polystyreneparticles after 24 h incubation with Staphylococcus epidermidis.

The biofilm colonization of the polystyrene particles in FIG. 7B) canreadily be observed by the fluffy, furry nature of the surface of theparticles compared to the smooth surface of the polystyrene particles inFIG. 7A). FIGS. 7C) and 7D) show the effect of the two peptide coatingson the colonization by Staphylococcus epidermidis. In particular,coating with Argininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl(FIG. 7C)) is markedly less effective than coating withArgininyl-(2,5,7-tri-tert-butyl)tryptophanyl-argininyl-aminohexanoyl(Figure d)).

Example 6 Biofilm Formation and Quantification of Activity AgainstBiofilms

Biofilm formation was induced in 96-well flat bottom microtitre plates(Nunclon Surface, NUNC). First, overnight cultures were diluted 1:100 inMHIIB (S. epidermidis and S. haemolyticus) or tryptic soy broth (TSB)with 5% glucose and 5% NaCl (S. aureus). 200 ml of this bacterialsuspension (107 cfu/ml) was added to each well and incubated for 24 h at37° C. After 24 h the wells were carefully washed twice withphosphate-buffered saline (PBS) to remove planktonic bacteria. Thewashing procedure was carefully evaluated by measuring metabolicactivity of the PBS with the Alamar blue method, described in detailbelow.

The washed biofilms were subjected to treatment with the Compounds atdifferent concentrations.

Trifluoroacetate salts of the compounds were dissolved in sterile waterand diluted to 5 mg/L, 50 mg/L and 500 mg/L in MHIIB. 200 μl of theCompounds, in different concentrations, were added to each well andincubated for 24 h at 37° C. Positive controls were untreated biofilmsonly added 200 μl MHIIB. Negative controls were only 200 μl MHIIB, withno bacteria added.

The metabolic activity of the biofilm was quantified with a slightlymodified method previously described by Pettit et al. Antimicrob. AgentsChemother. 2005; 49: 2612-7. Briefly, after the 24 h incubation withantimicrobial agents the wells were again washed twice with PBS and thenadded 250 ml MHIIB with 5% Alamar blue (AB; Biosource, Camarillo,Calif., USA). AB is a redox indicator which both fluoresces and changescolour in response to chemical reduction. The extent of reduction is areflection of bacterial cell viability. After 1 h incubation at 37° C.,absorbance was recorded at 570 and 600 nm using Versamax tuneablemicroplate reader (Molecular Devices, Sunnyvale, Calif., USA). Allassays were performed 3 times with 8 parallels. The highest and lowestvalue of each run was excluded from the analyses, and the remaining 18values were averaged.

Peptide Sequences

Peptide Sequence ME 143 R-Phe(4-(2′-naphthyl))-R—NH—CH(CH3)2

Minimum Inhibitory Concentration (MIC) on Planctonic Bacteria

MIC ME 143  8-16 S. epidermidis 32 μg/ml 42-77 S. epidermidis 32 μg/ml51-03 S. haemolyticus 16 μg/ml 51-07 S. haemolyticus 16 μg/ml PIA 9 S.aureus 64 μg/ml PIA 90 S. aureus 64 μg/ml

Minimum Biofilm Inhibitory Concentration (MBIC) for ME 143 Measured asSurvival Rate (%)

MIC 5 μg/ml 50 μg/ml 500 μg/ml  8-16 S. epidermidis 100 75 6 42-77 S.epidermidis 100 80 12 51-03 S. haemolyticus 100 20 9 51-07 S.haemolyticus 100 12 8 PIA 9 S. aureus 100 100 14 PIA 90 S. aureus 100 807

1-22. (canceled)
 23. A compound of formula (II):AA₁-AA₂-AA₃-R₁-R₂  (II) wherein AA₁ and AA₃ are independently lysine,arginine, histidine or a cationic analogue of lysine, arginine orhistidine, and AA₂ is tributyl tryptophan or a biphenylalaninederivative selected from the group consisting of Phe (4-(2′-naphthyl)),Phe (4-(1′-naphthyl)), Phe(4-4′-n-butylphenyl), Phe (4-4′-biphenyl) andPhe(4-4′-t-butylphenyl); R₁ is a N atom, which may be substituted by abranched or unbranched C₁-C₁₀ alkyl or aryl group, which group mayincorporate up to 2 heteroatoms selected from N, O and S; and R₂ is analiphatic moiety having 2-20 non-hydrogen atoms, said moiety beinglinear, branched or cyclic.
 24. The compound of claim 23, wherein saidcompound is a peptide.
 25. The compound of claim 23, wherein R₁ isunsubstituted.
 26. The compound of claim 23, wherein R₂ comprises 3 to 6non-hydrogen atoms.
 27. The compound of claim 23, wherein thenon-hydrogen atoms of R₂ are carbon atoms.
 28. The compound of claim 23,wherein R₂ is a linear or branched alkyl group.
 29. The compound ofclaim 28, wherein said alkyl group is selected from the group consistingof ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl and isomers thereof, and hexyl and isomers thereof.
 30. Thecompound of claim 23, wherein —R₁-R₂ together is selected from the groupconsisting of —NHCH(CH₃)₂, —NH(CH₂)₅CH₃, —NH(CH₂)₃CH₃, —NH(CH₂)₂CH₃,—NHCH₂CH(CH₃)₂, —NHcyclohexyl and —NHcyclopentyl.
 31. A solid supporthaving attached thereto the compound as claimed in claim
 23. 32. Amethod of treating a tumor in a subject, said method comprising theadministration to a subject of the compound of claim
 23. 33. A method ofinhibiting biofilm formation or removing a biofilm ex vivo, said methodcomprising contacting said biofilm with the compound of claim
 23. 34. Amethod of treating a bio-film associated infection in a subject, saidmethod comprising the administration to a subject of the compound ofclaim 23.